1. Field of the Invention
This invention relates to the production of novel synthetic peptide constructs and the use of such constructs as antigens in the production of antibodies, vaccines, antiviral agents, and the like. The antibodies produced in accordance with the invention may be used diagnostically or therapeutically. The constructs may also be used as vaccines.
Antibodies are important products of the immune system. An antibody is an immunoglobulin, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art. Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(abxe2x80x2)2 Gabxe2x80x2, and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular molecule is maintained.
Antiserum containing antibodies, usually referred to as polyclonal antibodies, is obtained by well-established techniques involving immunization of an animal, such as a rabbit, guinea pig, or goat, with an appropriate immunogen and obtaining antisera from the blood of the immunized animal after an appropriate waiting period. State-of-the-art reviews are provided by Parker, xe2x80x9cRadioimmunoassay of Biologically Active Compounds,xe2x80x9d Prentice-Hall (Englewood Cliffs, N.J., U.S., 1976); Butler, J. Immunol. Meth. (1975) 7: 1-24; Broughton and Strong, Clin. Chem. 22: (1976) 726-732; and Playfair, et al., Br. Med. Bull. (1974) 30: 24-31.
Antibodies can also be obtained by somatic cell hybridization techniques, such antibodies being commonly referred to as monoclonal antibodies. Monoclonal antibodies may be produced according to the standard techniques of Kxc3x6hler and Milstein, Nature (1975) 265:495-497. Reviews of monoclonal antibody techniques are found in xe2x80x9cLymphocyte Hybridomas,xe2x80x9d ed. Melchers, et al. Springer-Verlag (New York 1978), Nature (1977) 266:495; Science (1980) 208: 692, and Methods of Enzymology (1981) 73 (Part B):3-46. Samples of an appropriate immunogen preparation are injected into an animal such as a mouse and, after a sufficient time, the animal is sacrificed and spleen cells obtained. Alternatively, the spleen cells of a non-immunized animal can be sensitized to the immunogen in vitro. The spleen cell chromosomes encoding the base sequences for the desired immunoglobulins can be compressed by fusing the spleen cells, generally in the presence of a non-ionic detergent, for example, polyethylene glycol, with a myeloma cell line. The resulting cells, which include fused hybridomas, are allowed to grow in a selective medium, such as HAT-medium, and the surviving immortalized cells are grown in such medium using limiting dilution conditions. The cells are grown in a suitable container, e.g., microtiter wells, and the supernatant is screened for monoclonal antibodies having the desired specificity.
Various techniques exist for enhancing yields of monoclonal antibodies, such as injection of the hybridoma cells into the peritoneal cavity of a mammalian host, which accepts the cells, and harvesting the ascites fluid. Where an insufficient amount of the monoclonal antibody collects in the ascites fluid, the antibody is harvested from the blood of the host. Alternatively, the cell producing the desired antibody can be grown in a hollow fiber cell culture device or a spinner flask device, both of which are well known in the art. Various conventional ways exist for isolation and purification of the monoclonal antibodies from other proteins and other contaminants (see Kxc3x6hler and Milstein, supra).
In another approach for the preparation of antibodies the sequence coding for antibody binding sites can be excised from the chromosome DNA and inserted into a cloning vector which can be expressed in bacteria to produce recombinant proteins having the corresponding antibody binding sites.
Vaccines often comprise an antigen on a natural carrier such as a protein, a carbohydrate, a lipid or a liposome. Such vaccines are useful and have been employed for many years. There are, however, a number of art recognized problems with them. Several of these problems are related to the carrier. Since the carriers are isolated from natural sources, they are often not of uniform quality. Additionally, despite expensive and arduous purification efforts, it is difficult, and often impossible, to provide products completely free of natural contaminants. Such contaminants may themselves be antigenic. They cause the undesirable side reactions often associated with the use of vaccines, particularly fevers and tissue swelling. Additionally, the concentration of antigen may vary from one batch to another because the amounts of antigen, which react with the carrier or are absorbed on its surface are not uniform.
It is known that synthetic peptides can induce antibodies reactive with their cognate sequences in the native proteins. Specific antipeptide antibodies are useful laboratory reagents for confirming proteins from recombinant DNA, exploring biosynthetic pathways and precursors, and probing structural functions of proteins. Synthetic peptide antigens, conveniently available through chemical synthesis, can also be used for producing immunogens and for passive immunoprophylaxis.
One approach to preparing antipeptide antibodies is conjugation of a peptide to a known immunogenic carrier such as a protein. Examples of such carriers include albumins, serum proteins, e.g., globulins, ocular lens proteins and lipoproteins, bovine serum albumin, keyhole limpet hemocyanin (xe2x80x9cKLHxe2x80x9d), egg ovalbumin and bovine gamma-globulin. The peptide may also be linked to a synthetic polymer carrier or a liposome to give a macromolecular structure to the antigen carrier. Methods designed to avoid the use of carrier by polymerizing synthetic peptide antigens to give peptide polymers are also known. Although such materials are effective in producing animal antibodies, these materials are ambiguous in composition and structure. This shortcoming is particularly troublesome, for example, for antipeptide antibodies used for a human vaccine.
In an effort to address this problem, multiple antigen carrying structures were developed. Such structures are known and available commercially under the name Multiple Antigen Peptide System (MAPS). A small peptidyl core matrix is utilized bearing radially branching synthetic peptides as dendride arms. These molecules are produced by solid phase peptide synthesis beginning with three or four lysine residues which have only one kind of side chain-protecting group. Upon deprotection both amino groups are freed and a new similarly protected lysine derivative is condensed to the two free amino groups. This produces a branch chain with up to eight free amino groups which can then be used to synthesize directly onto the scaffolding the desired antigen which is usually a relatively short peptide. These structures are typically subject to steric crowding, presenting disadvantages for synthesis, which suppress yields and lead to solutions of dendritic polymers with variable numbers of side chains. The practical limit for the MAPS approach is to include about 4 to 8 side chains, typically presenting the identical antigen. Moreover, the relatively tight steric crowding of side chains can interfere with antigen presentation.
2. Previous Disclosures
A synthetic peptide vaccine design and the synthesis and properties of a high-density multiple antigenic peptide system is described by Tam in Proc. Natl. Acad. Sci. USA (1988) 85:5409-5413.
A multiple antigen peptide system is disclosed in U.S. Pat. No. 5,229,490 issued Jul. 20, 1993 (Tam I).
A multiple antigen peptide system having adjuvant properties, vaccines prepared therefrom and methods of use therefor are discussed in U.S. Pat. No. 5,580,563 issued Dec. 3, 1996 (Tam II).
Synthetic peptides and use thereof in preparing calmodulin antisera are disclosed in U.S. Pat. No. 4,716,150 issued Dec. 29, 1987 (Van Eldik).
Multiple antigen peptides for use as HIV vaccines is discussed in PCT application WO 93/03766 published on Mar. 4, 1993 (Tam III).
A leukemia-associated virus immunogen, vaccine and assay are disclosed in U.S. Pat. No. 4,794,168 issued Dec. 27, 1988 (Elder).
One aspect of the invention concerns a synthetic construct comprising a linear core chain having two or more side chains preferably including an epitopic site pending directly from different points on the linear core chain. Each of the side chains comprises an epitopic site of an antigen.
Another aspect of invention is a polymer comprising one or more of the above synthetic constructs linked together. In this embodiment the epitopic sites of the side chains or respective constructs may be the same or different.
Another embodiment of the present invention is a synthetic construct comprising a linear sequence of amino acids having two or more peptides having the same sequence pending directly from different points on the linear sequence. The invention also includes a polymer comprising one or more of the above synthetic constructs linked together wherein the peptides or respective constructs may be the same or different.
Another aspect of the present invention concerns a support having one or more of the above synthetic constructs or polymers coupled thereto.
Another embodiment of the present invention is directed to antibodies raised against the above synthetic constructs or polymers by administering the above synthetic constructs or polymers to a host. The antibodies can also include antibodies that are purified using the above mentioned supports.
Another aspect of the present invention is a vaccine comprising the above synthetic construct.
Another aspect of the present invention is a method of synthesizing the present polymers. In the method a synthetic construct comprising a linear core chain having two or more side chains pending directly from different points on the linear core chain is formed. Each of the side chains comprises an epitopic site of an antigen. Two or more of the synthetic constructs are then coupled together. In one aspect of the method each of the side chains on the linear core chain comprises a first reactive functionality. The synthetic construct is prepared by combining this functionalized linear core chain with a peptide having a second functionality reactive with the first reactive functionality.
Another embodiment of the present invention is a synthetic construct comprising a linear sequence of amino acids, at least one of which is lysine and having a peptide pending from the terminal amino group. In this embodiment the peptide may be further linked to the terminal amino group of the lysine by an aspartic acid molecule.
In a particular embodiment of the present invention the epitopic site of the antigen is the active site of human relaxin. The invention also includes an antibody raised against the synthetic construct wherein the epitopic site of the antigen is the active site of human relaxin and in particular relaxin-H2.